臺灣博碩士論文加值系統

Lon蛋白酶是一個單一聚合且多功能的酵素，它被高度的保存在生物種之間。Lon蛋白酶分為兩種亞型，LonA及LonB。它們保有ATPase domain歸屬於AAA+ superfamily之中並利用serine-lysine catalytic dyad 作為酵素的活性位置。LonB不同於LonA其具有N端的區段反而具穿膜區塊使之可鑲嵌在細胞膜上。Lon蛋白酶可調節生物體中新陳代謝的過程，適時降解目標蛋白以維持蛋白質的功能與結構的完整性。本篇論文選擇台灣本土嗜熱菌Meiothermus taiwanensis (WR-220)，選殖出兩個Lon蛋白酶MtaLonA1(793 a.a)、MtaLonA2(815 a.a)並建構其特異區塊酵素MtaLonA1N (1-312 a.a)、MtaLonA1A (313-585 a.a)、MtaLonA1α (492-585 a.a)、MtaLonA1C (586-793 a.a)、MtaLonA1NA (1-585 a.a)、MtaLonA1AC (313-793 a.a)、MtaLonA2N (1-320 a.a)、MtaLonA2A (321-601 a.a)、MtaLonA2α (508-601 a.a)、MtaLonA2C (602-815 a.a)、MtaLonA2NA (1-601 a.a)、 MtaLonA2AC (321-815 a.a)，透過表現與純化探討其結構與功能的特性，進而比較MtaLonA1及MtaLonA2的差異。
結構方面，利用原二色偏光儀分析呈現，所有蛋白皆以α-螺旋為主要的二級結構並且具有完整的三級結構。進一步利用AUC算得MtaLonA1及MtaLonA2分別形成六聚體及四聚體，於原態膠體電泳實驗中，MtaLonA1N、A1NA、MtaLonA2N、A2NA特異區塊酵素具有多聚體的結構。活性方面，MtaLonA1具有Protease、Peptidase、ATPase、Chaperone-like活性；然而，MtaLonA2具有Protease、ATPase、DNA-binding及較弱的Chaperone-like活性。在MtaLonA2的特異區塊酵素中，只有MtaLonA1N不具有DNA-binding的活性；MtaLonA2α呈現較弱的DNA-binding能力。尤其是MtLonA2AC呈現Protease、Peptidase活性及些許的ATPase活性。所有具有N端的蛋白(MtaLonA1N、A1NA、A2N、A2NA)皆有Chaperone-like活性，藉由此結果推斷出N端的序列對於結構聚合及Chaperone-like活性是必要的。我們並發現MtaHUβ為MtaLonA2特有的目標蛋白。基於這些實驗的結果，我們提出MtaLonA1及MtaLonA2具有不同的功能，更重要的是找出特異性的目標蛋白並結晶出完整長度的Lon蛋白酶，以深入了解MtaLonA1及MtaLonA2在生物體之中所參與的細胞調解。

ATP-dependent Lon protease has been known as one of the most evolutionarily conserved proteins in living organisms, which is a homo-oligomeric multi-domain enzyme. Lon proteases are divided into two subfamilies, LonA and LonB. Each possesses both ATPase domain, belonging to the AAA+ superfamily and a proteolytic domain (P-domain) with a serine-lysine catalytic dyad. The difference between LonA and LonB is that LonA contains a large N-terminal domain, whereas LonB has a transmembrane domain that anchors the protein to the membrane. Lon proteases are well-known to regulate the metabolic processes and involve in protein quality control system. In this study, we analyzed the primary sequence of MtaLonA1 (793 a.a) and MtaLonA2 (815 a.a) from Meiothermus taiwanensis (WR-220) and constructed the truncated-domain MtaLonA1N (1-312 a.a), MtaLonA1A (313-585 a.a), MtaLonA1α (492-585 a.a), MtaLonA1C (586-793 a.a), MtaLonA1NA (1-585 a.a), MtaLonA1AC (313-793 a.a), MtaLonA2N (1-320 a.a), MtaLonA2A (321-601 a.a), MtaLonA2α (508-601 a.a), MtaLonA2C (602-815 a.a), MtaLonA2NA (1-601 a.a) and MtaLonA2AC (321-815 a.a). MtaLonA1, MtaLonA2 and their truncated proteins are overexpressed and purified to examine the functional and structural properties. The structural characteristic presented by circular dichroism revealed that all constructs were composed of α-helical as the major secondary structures and all possessed well-defined three-dimensional structures. For quaternary structure, MtaLonA1 reveals mainly as hexamer and MtaLonA2 might be as tetramer. In native-PAGE, MtaLonA1N-, A1NA- and MtaLonA2N-, A2NA-domain exhibited polymeric structures. Functional studies demonstrated that MtaLonA1 shows the protease, peptidase, ATPase and chaperone-like activities; whereas MtaLonA2 shows the protease, ATPase, DNA-binding activity and weaker chaperone-like activities than that of MtaLonA1. Among the truncated proteins of MtaLonA2, only MtaLonA2N (N-domain) shows no DNA-binding activity, and the MtaLonA2α-domain) reveals weaker DNA-binding capability compared to MtaLonA1α. Particularly, MtaLonA2AC has peptidase activity, protease activity, but only slight ATPase activity. MtaLonA1N-, MtaLonA1NA-, MtaLonA2N- and MtaLonA2NA domain have chaperone-like activities. These results suggested that N-terminal sequence is essential for oligomerization and chaperone-like activities. We found out the substrate, MtaHU β is specific to the MtaLonA2. Based on these results, we propose that MtaLonA1 and MtaLonA2 may have different functions and it is important to find out the specific natural substrates and crystallize structures of full-length enzymes.

謝誌 vii
中文摘要 viii
Abstract x
1. Introduction 1
1.1 Lon protease 1
1.2 The structure of Lon protease 3
1.3 The function of Lon protease 4
1.4 The substrate of Lon protease 6
1.5 Thermophiles 9
1.6 The aim of this study 11
2. Materials and Methods 13
2.1 Sequence alignment 13
2.2 Cloning of MtaLonA1, MtaLonA2 and truncated proteins 13
Table 1. Oligonucleotides used in this study 15
2.3 Expression and purification of MtaLonA1, MtaLonA2 and truncated proteins 16
2.4 Circular Dichroism (CD) spectroscopy 17
2.5 Analytical Ultracentrifugation (AUC) 18
2.6 Native polyacrylamide gel electrophoresis 19
2.7 Transmission Electron Microscopy (TEM) 19
2.8 Dynamic Light Scattering (DLS) 20
2.9 Electrophoresis Mobility Shift Assay 20
2.10 Protease assay 21
2.11 Peptidase assay 22
2.12 ATPase assay 22
2.13 Chaperone activity assay 23
2.14 Phylogenetic analysis of Meiothermus taiwanensis LonA1 and LonA2 24
3. Results 26
3.1 Determination of MtaLonA1, MtaLonA2 and their truncated proteins from M. taiwanesis WR 220 26
Figure 1. Scheme of MtaLonA1, MtaLonA2 and their truncated form 27
Figure 2. Multiple alignments of amino acid sequences of MtaLonA1 and MtaLonA2 with other LonA protease 28
3.2 Expression and purification 29
Figure 3. SDS-PAGE of purified recombinant MtaLonA1, MtaLonA2 and their truncated proteins 30
3.3 Circular dichroism spectra of MtaLonA1, A2 and their truncated proteins 31
Figure 4. Far-ultraviolet circular dichroism spectra of MtaLonA1, MtalonA2 and their truncated proteins 33
Figure 5. Near-ultraviolet circular dichroism spectra of MtaLonA1, MtalonA2 and their truncated proteins-1 34
Figure 6. Near-ultraviolet circular dichroism spectra of MtaLonA1, MtalonA2 and their truncated proteins-2 35
3.4 Oligomerization of MtaLonA1, A2 and their truncated proteins 36
Figure 7. Sedimentation velocity experiment of MtaLonA1 and MtaLonA2 38
Figure 8. Sedimentation equibrium experiment of MtaLonA2 39
Figure 9. Quateruary structure of MtaLonA1, MtaLonA2 and their truncated proteins in Native PAGE 40
Figure 10. Transmission electron microscopy image of MtaLonA1 41
Figure 11. Transmission electron microscopy image of MtaLonA2 42
3.5 Characterization of proteolytic and ATPase activities of MtaLonA1, A2 and their truncated proteins 43
Figure 12. Effect of pH on protease activity of MtaLonA1 and MtaLonA2 45
Figure 13 Protease activity of MtaLonA1, MtaLonA2 and their truncated proteins 46
Figure 14. Peptidase activity of MtaLonA1, MtaLonA2 and their truncated proteins 47
Figure 15. ATPase activity of MtaLonA1, MtaLonA2 and their truncated proteins 48
3.6 Characterization of DNA-binding activity of MtaLonA1, A2 and their truncated proteins 49
Figure 16. Electrophoretic mobility shift assay of MtaLonA1 and their truncated proteins. 50
Figure 17. Electrophoretic mobility shift assay of MtaLonA2 and their truncated proteins. 51
3.7 Characterization of chaperone-like activity of MtaLonA1, A2 and their truncated proteins 52
Figure 18. Chaperone-like activity of MtaLonA1 S678A mutant and MtaLonA2 S694A mutant under chemical stress 55
Figure 19. Chaperone-like activity of MtaLonA1N and MtaLonA2N under chemical stress 56
Figure 20. Chaperone-like activity of MtaLonA1α under chemical stress 57
Figure 21. Chaperone-like activity of MtaLonA1C and MtaLonA2C under chemical stress 58
Figure 22. Chaperone-like activity of MtaLonA1NA and MtaLonA2NA under chemical stress 59
Figure 23. Chaperone-like activity of MtaLonA2AC under chemical stress 60
Figure 24. Chaperone-like activity of MtaLonA1 S678A mutant and MtaLonA2 S694A mutant under thermal stress 61
Figure 25. Refolding activity of MtaLonA1 S678A mutant and MtaLonA2 S694A mutant 62
Figure 26. Refolding activity of MtaLonA1 S678A mutant and MtaLonA2 S694A mutant 63
Figure 27. Refolding activity of MtaLonA1 and MtaLonA2 64
Figure 28. Refolding activity of MtaLonA1 and MtaLonA2 65
Figure 29. Refolding or proteolytic substrates by MtaLonA1 or MtaLonA2 66
3.8 Specific substrates of MtaLonA1 and MtaLonA2 67
Figure 30. Natural substrates of Lon protease in M. taiwanesis 68
4. Discussion 69
4.1 Phylogenetic analysis of MtaLonA1, MtaLonA2 69
Figure 31. Phylogenetic analysis of Lon proteases-1 71
Figure 32. Phylogenetic analysis of Lon proteases-2 72
Table 2. Materials of phylogenetic tree 73
Table 3. Materials of phylogenetic tree 74
4-2 Structural characteristics of MtaLonA1, MtaLonA2 and their truncated proteins 75
4-3 DNA-binding region of Lon protease 77
Figure 33. Alignment of MtaLonA1, MtaLonA2 and Bt-Lon α-domains 80
4-4 Catalytic activity of MtaLonA1, MtaLonA2 and their truncated proteins 81
4-5 Characterization of chaperone-like activity of MtaLonA1 mutant, MtaLonA2 mutant and their truncated proteins 82
4-6 Physiological roles of multi-functional MtaLonA1 and MtaLonA2 86
Reference 88
Appendix 98
Figure 1. Particle size of MtaLonA2 98

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